LncRNA RNA ROR Aggravates Hypoxia/Reoxygenation-Induced Cardiomyocyte Ferroptosis by Targeting miR-769-5p/CBX7 Axis.

Ferroptosis is a new way of cell death which is reported to participate in the pathology of myocardial ischemia-reperfusion (MI/R) injury, but it's mechanism remains unclear. The present investigation is to study the emerging role of long non-coding RNA (lncRNA) regulator of reprogramming (ROR) in cardiomyocyte ferroptosis after hypoxia/reoxygenation (H/R) administration. RT-qPCR and/or Western blot methods were performed to examine the gene/or protein levels, and CCK-8, ELISA, and DCFH-DA staining determined the cellular viability and ferroptosis. Dual-luciferase and RNA immunoprecipitation were applied to verify molecular interaction. LncRNA ROR and miR-769-5p were overexpressed and reduced in blood samples from MI patients and H/R-treated AC16 cells, respectively. Mechanistically, lncROR sponged to miR-769-5p, thus upregulating CBX7 expression. Functional experiments presented that lncRNA ROR silence mitigated H/R-stimulated inflammatory damage, oxidative stress, and ferroptosis in AC16 cells, whereas these roles could be reversed by co-downregulation of miR-769-5p or co-overexpression of CBX7. These data uncovered that lncRNA ROR prevented against H/R-induced cardiomyocyte ferroptosis by modulating miR-769-5p/CBX7 signaling, emphasizing the therapeutic value of lncRNA ROR in MI/R injury.

LncRNA ROR promotes NLRP3-mediated cardiomyocyte pyroptosis by upregulating FOXP1 via interactions with PTBP1.

OBJECTIVE: The purpose of this design was to explore the specific role and related mechanism of long noncoding RNA (lncRNA) regulators of reprogramming (ROR) in viral myocarditis (VMC). METHODS: AC16 cells were infected with coxsackievirus B3 (CVB3) to establish a VMC cell model in vitro. The release of interleukin (IL)-1ß and IL-18 was evaluated by enzyme-linked immunosorbent assay (ELISA). Gene expression was calculated using quantitative real-time (qRT)-PCR. Cell pyroptosis was determined by flow cytometry and Western blot assays. Cell counting Kit-8 (CCK-8) detected cell viability. The molecular associations were verified by employing RNA immunoprecipitation (RIP), RNA pulldown and chromatin immunoprecipitation (ChIP) assays. RESULTS: The lncRNA ROR was more highly expressed in CVB3 virus-infected AC16 cells than in controls. Knockdown of ROR markedly rescued cell viability and reduced the release of IL-1ß and IL-18, cell pyroptosis and pyroptotic proteins such as NLRP3, ASC and cleaved caspase 1. Mechanistically, ROR destroyed the mRNA stability of Forkhead Box P Factor 1 (FOXP1) by binding polypyrimidine tract binding protein 1 (PTBP1). FOXP1 repressed the transcription of NLRP3 by directly interacting with its promoter. Importantly, coinhibition of FOXP1 impeded the protective role of ROR silencing in CVB3-infected AC16 cells. CONCLUSION: In conclusion, these findings elucidated that ROR knockdown inhibited CVB3-induced cardiomyocyte inflammation and NLRP3-mediated pyroptosis by regulating the PTBP1/FOXP1 axis, implying that ROR might be a new inducer in CVB3-infected VMC.

Long non-coding RNA ROR sponges miR-138 to aggravate hypoxia/reoxygenation-induced cardiomyocyte apoptosis via upregulating Mst1.

BACKGROUND: Hypoxia/reoxygenation (H/R) injury of cardiomyocytes causes an irreversible damage to heart and largely results in acute myocardial infarction. Study has indicated lncRNA ROR aggravates myocardial ischemia/reperfusion (I/R) injury. Also, lncRNA ROR sponges miR-138 to promote osteogenesis. MiR-138 involves in hypoxic pulmonary vascular remodelling by targeting Mst1. However, the interaction between lncRNA ROR, miR-138 and Mst1 involved in myocardial H/R injury is still unknown. METHODS: H9C2 cells were used to establish H/R injury model. The expression levels of lncRNA ROR and miR-138 were modified by transfection with the miR-138 mimics or lncRNA ROR overexpression plasmid. MTT and flow cytometry analysis were performed to detect cell proliferation and apoptosis. Dual luciferase reporter assay was used to determine interaction between lncRNA ROR and miR-138 or miR-138 and Mst1. Expression levels of lncRNA ROR, miR-138, Mst1 and apoptosis-related markers were determined by qRT-PCR or western blotting. RESULTS: LncRNA ROR was significantly up-regulated, while miR-138 was obviously down-regulated in H/R-induced injury of H9C2 cells. Furthermore, miR-138 overexpression alleviated cardiac cell apoptosis induced by H/R injury. Mst1 was revealed to be a target of miR-138 and negatively regulated by miR-138. Mst1 overexpression reversed the protective effects of miR-138 on H/R injury of H9C2 cells. LncRNA ROR was identified as a sponge for miR-138. MiR-138 could protect H9C2 cells form H/R injury induced by lncRNA ROR overexpression. CONCLUSION: Our study provides that lncRNA ROR sponges miR-138 to aggravate H/R-induced myocardial cell injury by upregulating the expression of Mst1.

LncRNA-ROR alleviates hypoxia-triggered damages by downregulating miR-145 in rat cardiomyocytes H9c2 cells.

Chronic hypoxic heart disease (CHD) is a common clinical type of congenital heart disease. Long noncoding RNA regulator of reprogramming (lncRNA-ROR) exerts an important regulating effect in cardiovascular diseases. In our study, we explored the effect of lncRNA-ROR and the possible mechanisms against hypoxia-caused apoptosis in H9c2 cells. H9c2 cells were exposed to hypoxia (1% O2 ) to construct the in vitro model of CHD. The level of lncRNA-ROR and microRNA (miRNA/miR)-145 was detected. To upregulate the level of lncRNA-ROR and miR-145, transfection was carried out. Western blot assay was performed to quantified protein expression. The interaction of lncRNA-ROR with miR-145 was verified by RIP and Dual-luciferase reporter assays. The expression of p53 and Bax was largely elevated and Bcl-2 was suppressed by hypoxia induction. We found that lncRNA-ROR was elevated by hypoxia. LncRNA-ROR overexpression was able to relieve the damages of H9c2 cells induced by hypoxia through rescuing viability, suppressing apoptosis, and blocking Cytochrome c release. miR-145 was suppressed by overexpressed lncRNA-ROR and the combination of miR-145 mimic was able to abolish the protective effect of lncRNA-ROR. Moreover, we found that lncRNA-ROR activated Ras/Raf/MEK/ERK and PI3K/AKT transduction cascades by suppressing miR-145. Besides, lncRNA-ROR directly targeted miR-145 and negatively modulated the level of miR-145. Our present study revealed that lncRNA-ROR protected H9c2 cells against hypoxia-caused damages by regulation of miR-145 through activating Ras/Raf/MEK/ERK and PI3K/AKT.

The lncRNA ROR/miR-124-3p/TRAF6 axis regulated the ischaemia reperfusion injury-induced inflammatory response in human cardiac myocytes.

Myocardial ischaemia reperfusion injury (MIRI) is considered the primary cause of death in patients with cardiovascular diseases. Recently, long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been found to be involved in the pathogenesis of MIRI. However, whether lncRNA ROR and miR-124-3p play roles in MIRI and the underlying mechanism remain undetermined. HCMs were exposed to hypoxic conditions for 2 h followed by re-oxygenation (H/R) treatment. Expression of miR-124-3p and lncRNA ROR in HCMs was measured by qRT-PCR. TRAF6 expression was evaluated by qRT-PCR and western blotting. ELISA and qRT-PCR were conducted to assess the production of TNF-α, IL-6, and IL-1ß. The interaction between miR-124-3p and TRAF6, as well as between miR-124-3p and lncRNA ROR, was verified by dual-luciferase reporter assay. Cell apoptosis was detected by flow cytometry analysis. Our data revealed that miR-124-3p was significantly downregulated, while TRAF6 and lncRNA ROR were upregulated in both MIRI rat model and H/R treated HCMs. Overexpression of miR-124-3p reversed the H/R-induced cell apoptosis and upregulation of TNF-α, IL-6, and IL-1ß. Mechanistically, miR-124-3p bound and negatively regulated TRAF6 expression in HCMs. Moreover, TRAF6 overexpression significantly blocked the effects of miR-124-3p mimics on cell apoptosis and inflammatory response of HCMs, which involved the NF-κB pathway. Further analysis showed that lncRNA ROR sponged and negatively regulated miR-124-3p in HCMs. Overexpression of IL-1ß was demonstrated to promote H/R induced cell apoptosis in HCMs. In addition, overexpression of ROR further enhanced the H/R-induced inflammation and cell apoptosis through its action on miR-124-3p. The lncRNA ROR/miR-124-3p/TRAF6 axis regulated the H/R-induced cell apoptosis and inflammatory response of HCMs.

LncROR Promotes Bladder Cancer Cell Proliferation, Migration, and Epithelial-Mesenchymal Transition.

BACKGROUND: LncRNA ROR, a tumor oncogene associated with various human cancers, has been reported to be involved in regulating various cellular processes, such as proliferation, apoptosis and invasion through targeting multiple genes. However, the molecular biological function in bladder cancer has not been clearly elucidated. The aim of this study is to explore ROR expression levels and evaluated its function in bladder cancer. METHODS: LncRNA ROR expression levels in the 36 pairs of bladder cancer tissues (and corresponding non-tumor tissues) and bladder cancer cells were assessed by qRT-PCR. MTT assay, colony formation assay, flow cytometric analysis, wound healing assay, cell transwell assays, attachment/detachment and western blotting were performed to assess the effects of ROR on proliferation, apoptosis, migration/invasion and epithelial-to-mesenchymal (EMT) phenotypes in BC cells in vitro. ZEB1 is target of ROR. Rescue assays were performed to further confirm that ROR contributes to the progression of BC cells through targeting ZEB1. RESULTS: LncRNA ROR was up-regulated in bladder cancer tissues (compared to adjacent non-tumor tissues) and was almost overexpression in bladder cancer cells (compared with normal urothelial cell line SVHUC-1 cells). Increased lncRNA ROR expression significantly promoted tumor cells proliferation, inhibited cells apoptosis, facilitated cells metastasis and contributed to the formation of EMT phenotype. While down-regulated ROR could obviously inhibit cells proliferation, promote cells apoptosis, inhibit metastasis and reverse EMT to MET. ZEB1 was a target gene of ROR and was positive correlation with the level of ROR in cancer tissues. CONCLUSION: These results indicated that lncRNA ROR was associated with tumor progression in bladder cancer cells.

Long non-coding RNA ROR promotes proliferation, migration and chemoresistance of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is one of the most common malignancies of the head and neck. It arises from the nasopharynx epithelium and is associated with high morbidity and mortality. Long non-coding RNA (lncRNA) have been reported to regulate gene interaction and play critical roles in carcinogenesis and progression. LncRNA-ROR, a recently identified lncRNA, has been shown to be involved in initiation, progression and metastasis of several tumors, including hepatocellular carcinoma, breast cancer and glioma. However, whether lncRNA-ROR is associated with the progression of NPC remains unknown. Resistance to radiotherapy and chemotherapy is the primary cause of NPC patients' death. In this study, we found that lncRNA-ROR was significantly upregulated in NPC tissues compared with normal tissues. Next, our study proved that lncRNA-ROR was highly associated with the proliferation, metastasis and apoptosis of NPC. The enrichment of lncRNA-ROR played a critucal functional role in chemoresistance. The mechanism by which NPC resists chemotherapy might be that lncRNA-ROR suppress p53 signal pathway. Taken together, these data suggested that lncRNA-ROR played an important role in the progression of NPC; thereby it might become a therapeutic target and reduce chemoresistance for NPC.

Overexpression of LncRNA-ROR predicts a poor outcome in gallbladder cancer patients and promotes the tumor cells proliferation, migration, and invasion.

LncRNA-ROR has been reported to be involved in many kinds of human cancers. However, whether LncRNA-ROR is involved in gallbladder cancer progression remains largely unknown. The objective of this study is to investigate the role of LncRNA-ROR in gallbladder cancer. We found that LncRNA-ROR expression level was upregulated in gallbladder cancer tissues (P < 0.05) and was significantly associated with tumor sizes (P < 0.05) and lymph node metastasis (P < 0.05). High expression of LncRNA-ROR was significantly associated with poor prognosis in gallbladder cancer patients (P < 0.05). Moreover, knockdown of LncRNA-ROR inhibited cell proliferation, migration, and invasion. The epithelial-mesenchymal transition (EMT) phenotype induced by TGF-ß1 was reversed after LncRNA-ROR knocking down in SGC-996 and Noz cells. LncRNA-ROR plays an important role in the development of gallbladder cancer and mediates the EMT in gallbladder cancer. LncRNA-ROR might act as a marker of prognosis and therapeutic target for gallbladder cancer.

Exosomal lncRNA ROR1-AS1 from cancer-associated fibroblasts inhibits ferroptosis of lung cancer cells through the IGF2BP1/SLC7A11 signal axis.

BACKGROUND: Targeting ferroptosis is a potential strategy for cancer treatment. Activated cancer-associated fibroblasts (CAFs) can affect the progression of lung cancer through exosomes. This study investigated the mechanism by which exosomal lncRNA ROR1-AS1 derived from CAFs affects ferroptosis of lung cancer cells. METHODS: CAFs were identified by western blot and immunofluorescence. Exosomes derived from CAFs (CAF-exo) were analyzed by transmission electron microscope, nanoparticle tracking analysis and western blot. The expression levels of ROR1-AS1, IGF2BP1 and SLC7A11 in lung cancer were analyzed by bioinformatics analysis and detected by qPCR and western blot. The lung cancer cells were treated with Erastin and/or CAF-exo, then cell viability was detected by cell counting kit-8, and the ferroptosis-related indicators were detected by corresponding kits. The relationship between IGF2BP1 and ROR1-AS1 or SLC7A11 was determined by RNA pull down and RNA immunoprecipitation, and their effects on cell ferroptosis were confirmed by rescue experiments. Xenotransplantation experiment was used to determine the effect of CAF-exo on tumor growth and ferroptosis in vivo. Immunohistochemistry was used to identify the Ki-67 and 4-HNE expression. RESULTS: ROR1-AS1, IGF2BP1 and SLC7A11 were upregulated in lung cancer and indicated poor prognosis. LncRNA ROR1-AS1 increased the stability of SLC7A11 mRNA by interacting with IGF2BP1. Exosomal ROR1-AS1 from CAFs inhibited ferroptosis of lung cancer cells in vitro and in vivo. The effect of ROR1-AS1 overexpression or IGF2BP1 overexpression on ferroptosis of lung cancer cells was partially reversed by IGF2BP1 silencing or SLC7A11 inhibition. CONCLUSIONS: CAFs secrete exosomal ROR1-AS1 to promote the expression of SLC7A11 by interacting with IGF2BP1, thereby inhibiting ferroptosis of lung cancer cells.

Long Noncoding RNA Regulator of Reprogramming Regulates Cell Growth, Metastasis, and Cisplatin Resistance in Gastric Cancer via miR-519d-3p/HMGA2 Axis.

Background: Gastric cancer (GC) is a common tumor found worldwide, and cisplatin is the first-line agent for the treatment of GC. However, the resistance to cisplatin is an obstacle. Here, we explored the biological mechanism of long noncoding RNA regulator of reprogramming (ROR) in the cisplatin resistance of GC. Materials and Methods: ROR, miR-519d-3p, and high mobility group protein A2 (HMGA2) expression in GC tissues and cells were measured by quantitative real-time polymerase chain reaction and Western blot. Cell viability, migration, invasion, and apoptosis were detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, transwell assay, and flow cytometry, respectively. The relative protein expression was detected by Western blot. The interactions between miR-519d-3p and ROR, HMGA2 were predicted using miRcode and starBase v2.0 online database, and then verified by dual luciferase reporter assay and RNA immunoprecipitation assay. In addition, the xenograft tumor mouse model was constructed to verify the biological role of ROR in vivo. Results: The levels of ROR, HMGA2 were significantly upregulated, and miR-519d-3p was apparently downregulated in GC tissues and cells. The miRcode and starBase v2.0 online websites and dual luciferase reporter assay validated that miR-519d-3p directly interacted with ROR and HMGA2. Furthermore, ROR knockdown downregulated HMGA2 to restrain cell proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), and cisplatin resistance in GC cells by targeting miR-519d-3p. In addition, the depletion of ROR repressed the xenograft tumor growth in vivo. Conclusion: In conclusion, we first found the ROR/miR-519d-3p/HMGA2 regulatory network to regulate cell proliferation, migration, invasion, EMT, and cisplatin resistance in GC, and this may shed light on the GC tumorigenesis.

Down-regulated lncRNA ROR in tumor-educated platelets as a liquid-biopsy biomarker for nasopharyngeal carcinoma.

PURPOSES: To evaluate the diagnostic value of tumor-educated platelets (TEP) lncRNA ROR for nasopharyngeal carcinoma (NPC). METHODS: Quantitative real-time PCR was used to determine the expression level of TEP lncRNA ROR in NPC patients (n = 50) as compared to normal subjects (n = 33). The ROC curve analysis was performed to assess the diagnostic value of TEP lncRNA ROR for NPC. Correlations between TEP lncRNA ROR and clinical parameters were further analyzed. RESULTS: The median of TEP lncRNA ROR was significantly lower in NPC patients than that in normal subjects (0.0209 vs 0.0610, p = 0.0019), while no significant difference was found in plasma lncRNA ROR. ROC analysis showed that TEP lncRNA ROR had a sensitivity of 60%, specificity of 70%, and accuracy of 63.9% in diagnosing NPC, and the area under ROC curve (AUC) was 0.70. The expression level of TEP lncRNA ROR in NPC showed no significant difference among different TNM stages. However, low level of TEP lncRNA ROR correlated well with positive Epstein-Barr virus (EBV) DNA (kappa value = 0.314, p = 0.06), TEP lncRNA ROR and EBV DNA had similar diagnostic positive rate (58.3%) for NPC, and the combination of TEP lncRNA ROR and EBV DNA increased the positive rate to 74%. CONCLUSION: The expression level of TEP lncRNA ROR was down-regulated in NPC and the diagnostic value of TEP lncRNA ROR was similar to EBV DNA. Our study indicated that TEP lncRNA ROR might serve as a novel type of liquid biopsy biomarker in diagnosis of NPC patients.

Apatinib Inhibits Stem Properties and Malignant Biological Behaviors of Breast Cancer Stem Cells by Blocking Wnt/ß-catenin Signal Pathway through Downregulating LncRNA ROR.

BACKGROUND: Cancer stem cells could influence tumor recurrence and metastasis. OBJECTIVE: To develop a new effective treatment modality targeting breast cancer stem cells (BCSCs) and to explore the role of Apatinib in BCSCs. METHODS: BCSCs were isolated from MDA-MB-231 cells by the immune magnetic beads method. BCSCs were treated with Apatinib, lentiviral plasmids (lncRNA ROR), and iCRT-3 (Wnt pathway inhibitors). Viability, colony numbers, sphere numbers, apoptosis, migration, invasion of BCSCs were detected by MTT, colony formation, tumorsphere, flow cytometry, wound-healing, transwell assays, respectively. The expressions of markers (ABCG2, CD44, CD90, and CD24), epithelial-mesenchymal transition (EMT)-related molecules (Ecadherin, N-cadherin, Vimentin, MMP-2, MMP-9), and Wnt/ß-catenin pathway-related proteins (Wnt3a, Wnt5a, ß-catenin) in breast cancer stem cells were determined by performing Western blot and qRT-PCR analysis. RESULTS: Apatinib decreased the viability and colony numbers of BCSCs in a concentration-dependent manner, and it also reduced sphere numbers, suppressed migration, invasion and lncRNA ROR expression, and induced apoptosis of BCSCs. However, these results were partially reversed by lncRNA ROR overexpression. Apatinib suppressed stem property, EMT process, and Wnt/ß-catenin pathway in BCSCs, which was partially reversed by lncRNA ROR overexpression. Moreover, lncRNA ROR overexpression increased the colony and sphere numbers and promoted the cell viability, apoptosis inhibition, migration, and invasion of BCSCs, but these effects were partially reversed by iCRT-3. LncRNA ROR overexpression increased the stem property, EMT process, and Wnt/ß-catenin pathway, which were partially counteracted by iCRT-3. CONCLUSION: Apatinib inhibited stem property and malignant biological behaviors of BCSCs by blocking the Wnt/ß-catenin signal pathway through down-regulating lncRNA ROR.

Salvianolic acid B suppresses hepatic stellate cell activation and liver fibrosis by inhibiting the NF-κB signaling pathway via miR-6499-3p/LncRNA-ROR.

BACKGROUND: Long non-coding RNA (LncRNAs) have been reported to play an important role in liver fibrosis and are closely associated with hepatic stellate cell (HSC) activation. We previously found that salvianolic acid B (Sal B) improves liver fibrosis by regulating the NF-κB signaling pathway. However, whether the LncRNA, regulator of reprogramming (LncRNA-ROR) plays a role in Sal B-mediated anti-fibrosis effects via the NF-κB signaling pathway remain unclear. PURPOSE: This study aimed to evaluate the effects of Sal B on HSC activation and liver fibrosis and investigate its mechanism from the perspective of LncRNA-ROR-mediated NF-κB signaling pathways. METHODS: LX-2 and T6 cell lines were cultured. Animal models of liver fibrosis were established using CCl4 in male BALB/c mice. Primary HSCs were isolated from mice and cultured. Serum biochemical and liver histological analyses were performed to evaluate the effects of Sal B on liver fibrosis. The index of HSC activation and the expression of LncRNA-ROR, microRNAs (miRNAs), and inflammatory factors were determined by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) or immunofluorescence staining. Cell proliferation was measured by a Cell Counting Kit-8 (CCK-8). NF-κB signaling-associated protein levels were assessed using western blotting or immunofluorescence staining. A luciferase reporter assay was used to detect transcription activity. RESULTS: In this study, a lower level of LncRNA-ROR was found during Sal B attenuating HSC activation in HSCs. Mechanistically, Sal B impeded the NF-κB signaling pathway to inhibit HSC proliferation and activation by downregulating LncRNA-ROR. Additionally, Sal B upregulated miR-6499-3p to target LncRNA-ROR for degradation. Functionally, Sal B treatment ameliorated CCl4-induced liver fibrosis in mice by inhibiting HSC activation. CONCLUSION: Sal B suppresses HSC activation and liver fibrosis via regulation of miR-6499-3p/LncRNA-ROR-mediated NF-κB signaling pathway. These results reveal a new molecular mechanism of Sal B on liver fibrosis from the insight of LncRNAs.

LncRNA ROR/miR-145-5p axis modulates the osteoblasts proliferation and apoptosis in osteoporosis.

Osteoporosis (OP) is a systemic bone metabolic disease. Promotion of osteoblast proliferation and inhibition of cell apoptosis may be helpful for the prevention and clinical treatment of OP. In the current study, we focused on the expression changes and clinical values of lncRNA ROR and miR-145-5p in OP clinical serum samples, and investigated the interactive modulation effect of ROR/miR-145-5p on osteoblast function. Serum samples were obtained from 82 OP patients and 79 healthy individuals. MC3T3-E1 was applied for the cell experiments. Levels of lncRNA ROR and miR-145-5p were detected using qRT-PCR. Transient transfection was performed to regulate gene levels in cells, and cell proliferation and apoptosis were detected. A reciprocal correlation between lncRNA ROR and miR-145-5p was explored. LncRNA ROR was downregulated, and miR-145-5p was overexpressed in OP patients. The combined diagnosis of ROR and miR-145-5p showed good diagnostic value for OP. ROR knockdown promoted the MC3T3-E1 cell apoptosis and inhibited cell proliferation. Luciferase reporting assay verified the target relationship between ROR and miR-145-5p. MiR-145-5p downregulation reversed ROR silence mediated effect on MC3T3-E1 cell proliferation and apoptosis. LncRNA ROR is downregulated and miR-145-5p is highly expressed in OP patients. ROR knockdown may inhibit osteoblast proliferation via targeting miR-145-5p. It may provide a theoretical basis and experimental basis for ROR to be a potential target for the treatment of OP.

Ad36 promotes differentiation of hADSCs into brown adipocytes by up-regulating LncRNA ROR.

AIMS: This study is to investigate the role of adenovirus type 36 (Ad36) in inducing differentiation of human adipose-derived stem cells (hADSCs) into brown adipocytes. MAIN METHODS: The hADSCs were induced to differentiate into adipocytes by a cocktail method and Ad36, respectively. They were collected on the 2nd, 4th, 6th, and 8th day, respectively. LncRNA ROR was silenced by siRNA. RT-qPCR and Western-blot were used to detect the mRNA and protein levels. Transmission electron microscopy was used to observe the mitochondria. KEY FINDINGS: The mRNA and protein expression levels of LncRNA ROR, Cidea, Dio2, Fgf21, Ucp1, Prdm16, Cox5b, Atp5o, Atp6, and Nd2 in the Ad36 induction group were significantly higher than those in the cocktail induction group. The expression levels of Leptin mRNA and protein in the Ad36 induction group were significantly lower than those in the cocktail induction group. After siRNA knockdown of LncRNA ROR, mRNA and protein expression levels of Cidea, Dio2, Fgf21, Ucp1, Prdm16, Cox5b, Atp5o, Atp6 and Nd2 were significantly lower than the control group during the induction of hADSC differentiation into adipocytes by Ad36. Additionally, mitochondria in the Ad36 induction group was increased compared to that in the cocktail induction group. SIGNIFICANCE: Ad36 may promote the differentiation of hADSCs into brown adipocytes by up-regulating LncRNA ROR.

LncRNA ROR1­AS1 high expression and its prognostic significance in liver cancer.

Hepatocellular carcinoma (HCC) is a common disease of the digestive system with no curative treatments. Long noncoding RNA tyrosine protein kinase transmembrane receptor 1 antisense RNA 1 (lncRNA ROR1­AS1) is an lncRNA whose functions have been predicted in human diseases; however, its important role in cancer has been probed only in mantle cell lymphoma, not in HCC. Therefore, the present study aimed to elucidate the prognostic significance of lncRNA ROR1­AS1 in HCC. The Cancer Genome Atlas Liver Hepatocellular Carcinoma was used to analyze the expression of ROR1­AS1 in liver cancer. χ2 tests were performed to evaluate associations between clinical characteristics and ROR1­AS1 expression. The role of ROR1­AS1 in HCC prognosis was assessed using Kaplan­Meier curves and proportional hazards model (Cox) analysis. Gene set enrichment analysis was performed by using a Gene Expression Omnibus dataset. At the same time, Multi Experiment Matrix was used to predict genes that may be co­expressed with ROR1­AS1. The Database for Annotation, Visualization and Integrated Discovery and KO­Based Annotation System were used to analyze the most closely associated cytological behaviors and pathways in HCC. Then, the genes in the three databases were integrated to screen mRNAs, microRNAs and lncRNAs that had co­expression relationships with ROR1­AS1. Cytoscape, Search Tool for the Retrieval of Interacting Genes/Proteins and Molecular Evolutionary Genetics Analysis were used to map potential regulatory networks and developmental relationships associated with ROR1­AS1. Finally, 12 genes most closely associated with ROR1­AS1 were identified, and their relationship was described using a Circos plot. The results showed that ROR1­AS1 was upregulated in HCC, and its expression was related to clinical stage, T stage and N stage. Furthermore, Kaplan­Meier curves and Cox analysis indicated that high expression of ROR1­AS1 was associated with poor prognosis, and that ROR1­AS1 was an independent risk factor for HCC. Co­expression data suggested that there may be a large regulatory network of 45 genes with indirect associations with ROR1­AS1, a small regulatory network of 15 genes with direct or indirect regulatory relationships, and a special regulatory network containing 12 genes directly associated with ROR1­AS1. The present findings indicated that high expression of ROR1­AS1 suggests poor prognosis in patients with HCC.

Exosomal lncRNA ROR1-AS1 Derived from Tumor Cells Promotes Glioma Progression via Regulating miR-4686.

OBJECTIVE: Glioma is one of the most common central nervous system malignant tumors, accounting for 45%-60% of adult intracranial tumors. However, the clinical treatment of glioma is limited. It is of great significance to seek new therapeutic methods for glioma via gene therapy. MATERIALS AND METHODS: Microarray is used to identify the lncRNAs that are differentially expressed in glioma. The expression of long non-coding RNA (lncRNA) ROR1-AS1 and miR-4686 was detected by qRT-PCR. Exosomes were isolated from the supernatant of normal and cancerous cells, and TEM was used for exosomes identification. MTT assay, wound healing assay, transwell assay, and colony formation assay were used to detect the exo-ROR1-AS1 function on proliferation, migration, and invasion in glioma cells. Luciferase assay and RIP assay were used to identify the relationship between lncRNA ROR1-AS1 and miR-4686. The effect of exo-ROR1-AS1 on tumorigenesis of glioma was confirmed by the xenograft nude mice model. RESULTS: ROR1-AS1 was up-regulated in glioma tissues, and the high expression of ROR1-AS1 indicated a poor prognosis in glioma patients. Interestingly, ROR1-AS1 was packaged into exosomes and derived from tumor cells. Functional analysis showed exo-ROR1-AS1 promoted the progression of glioma cell lines SHG44 and U251. Furthermore, ROR1-AS1 acted as a sponge of miR-4686 and inhibited its expression. Functionally, forced expression of miR-4686 removed the promoted effects of lncRNA ROR1-AS1 on glioma development. In vivo tumorigenesis experiments showed that exo-ROR1-AS1 promoted glioma development via miR-4686 axis. CONCLUSION: Our study suggested tumor cells derived exo-ROR1-AS1 promoted glioma progression by inhibiting miR-4686, which might be a potential therapeutic target for glioma clinical treatment.

WDR7 up-regulation upon knocking down of neighboring non-coding RNA using siRNAs encapsulated in polyamidoamine dendrimers.

OBJECTIVES: Breast cancer is the second leading cause of cancer death in females. Understanding molecular mechanisms in cancer cells compared with normal cells is crucial for diagnostic and therapeutic strategies. Long intergenic non-protein coding RNA, a regulator of reprogramming (lincRNA-RoR) is a noncoding RNA which initially was detected in induced pluripotent stem cells, and it has an important role in cell reprogramming and highly expressed in breast cancer cells. A key point in successful gene silencing is the usage of siRNA delivery system that is safe and efficient. MATERIALS AND METHODS: In this study, the fifth-generation of PAMAM dendrimer is used as a nanocarrier for entering siRNA molecules for gene silencing of lincRNA-RoR. WDR7 is the gene encoding adjacent of lincRNA-RoR, which has an important role in apoptosis and cell cycle. Gel retardation assay was used to find the best Negative/Positive (N/P) molar charge ratio of siRNA- PAMAM transfected into MDA-MB 231 cells. MTT assay was performed 24 hr after transfection revealed the IC50 value (half maximal inhibitory concentrations) about 100 nanomolar for lincRNA-ROR siRNA. RESULTS: The lincRNA-RoR and WDR7 gene expression changes were evaluated by real-time PCR after siRNA treatment and showed an increase in the gene expression of WDR7. CONCLUSION: This study showed that PAMAM dendrimer G5/ siRNA could be a useful system delivery for future gene therapy approaches.

Long non-coding RNA ROR mitigates cobalt chloride-induced hypoxia injury through regulation of miR-145.

Obstructive sleep apnoea-hypopnoea syndrome (OSAHS) is a condition causing apnea and hypopnea. LncRNA-ROR revealed properties in regulating hypoxia response. Our study explored the roles of ROR in CoCl2-induced hypoxia injury in HK-2 cells. HK-2 cells were treated with CoCl2 to induce hypoxia injury. Cell viability, cell apoptosis and apoptotic proteins were detected using CCK-8, flow cytometry and western blot, respectively. The alter expression of ROR and miR-145 was achieved through transfection. Moreover, the expressions of HIF-α and ERK and MAPK related factors were examined using western blot. We found that CoCl2 decreased cell viability and increased apoptosis as well as increased the expression of ROR. ROR overexpression increased cell viability, decreased cell apoptosis. ROR overexpression upregulated anti-apoptotic proteins Bcl-2 and decreased p53, Bax and cleaved-Caspase-3. ROR overexpression also increased the expression of HIF-α. On the opposite, ROR silence led to the opposite results as relative to ROR overexpression. ROR overexpression decreased expression of miR-145. Co-transfection with ROR overexpression and miR-145 impaired the promoting effects of ROR in CoCl2 treated cells. ROR increased phosphorylation of ERK while decreased phosphorylation of MAPK. In conclusion, lncRNA ROR alleviated CoCl2-induced hypoxia injury through regulation of miR-145 as well as modulating ERK and MAPK signalling. Highlights CoCl2 induces ROR upregulation; Overexpression of ROR reduces CoCl2-induced HK-2 cell injury; Silence of ROR promotes CoCl2-induced HK-2 cell injury; Overexpression of ROR decreases miR-145 expression; ROR overexpression modulates ERK and MAPK signalling pathways through regulation of miR-145.

LncRNA ROR is involved in cerebral hypoxia/reoxygenation-induced injury in PC12 cells via regulating miR-135a-5p/ROCK1/2.

Ischemic stroke is a common cerebrovascular disease with high morbidity, disability and mortality. LncRNAs were involved in ischemia/reperfusion injury. The present study aims to investigate whether lncRNA ROR can promote the cerebral hypoxia/reoxygenation (H/R) injury in vitro, a cellular model of cerebral ischemia/reperfusion injury, through inhibiting the expression of miR-135a-5p or upregulating the expression of ROCK1 and ROCK2. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the lncRNA ROR expression in PC12 cells induced by H/R and verify the transfection effect. ROS, LDH, SOD and MDA levels were detected by respective kits. CCK-8 assay and flow cytometry analysis respectively detected the cell viability and cell apoptosis. Western blot analysis was to analyze the expression of apoptosis-related proteins (Bcl-2, Bax and cleaved caspase3). Immunofluorescent staining detected the ROCK1/2 expression. As a result, lncRNA ROR expression was increased in the PC12 cells induced by H/R. LncRNA ROR overexpression could aggravate injury of PC12 cells induced by H/R. And, lncRNA ROR overexpression could decrease viability and promote apoptosis of PC12 cells induced by H/R. In addition, miR-135a-5p was demonstrated to be a target of lncRNA ROR and lncRNA ROR improved H/R injury in PC12 cells by up-regulating the expression of miR-135a-5p via down-regulating ROCK1/2 expression. In conclusion, this study indicated that lncRNA ROR could promote the cerebral H/R injury by inhibiting the expression of miR-135a-5p or upregulating the expression of ROCK1/2. And, miR-135a-5p overexpression could improve the cerebral H/R injury by inhibiting the expression of ROCK1/2.